The mannose transporter complex acts by a mechanism which couples translocation with phosphorylation of the substrate. It consists of a hydrophilic subunit (IIAB) and two transmembrane subunits (IIC, IID). The purified complex was reconstituted into phospholipid vesicles by octyl glucoside dilution. Glucose export was measured with proteoliposomes which were loaded with radiolabeled glucose and to which purified IIAB, cytoplasmic phosphorylcarrier proteins, and P-enolpyruvate were added from the outside. Vectorial transport was accompanied by stoichiometric phosphorylation of the transported sugar. Glucose added to the outside of the proteoliposomes was also phosphorylated rapidly but did not compete with vectorial export and phosphorylation of internal glucose. Glucose uptake was measured with proteoliposomes which were loaded with the cytoplasmic phosphoryl carrier proteins and P-enolpyruvate and to which glucose was added from the outside. Vectorial import and phosphorylation occurred with a higher specificity (K 30 ± 6 µM, k 401 ± 32 pmol of Glc/µg of IICD/min) than nonvectorial phosphorylation (K 201 ± 43 µM, k 975 ± 88 pmol of Glc/µg of IICD/min).

A new plasmid pTSHIC9 for the controlled overexpression of the cytoplasmic phosphoryl carrier proteins, enzyme I, HPr, and IIA, and a simplified procedure for the purification of these proteins are also described.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. Hu, K. Hu, D. C. Williams Jr., M. E. Komlosh, M. Cai, and G. M. Clore Solution NMR Structures of Productive and Non-productive Complexes between the A and B Domains of the Cytoplasmic Subunit of the Mannose Transporter of the Escherichia coli Phosphotransferase System J. Biol. Chem., April 18, 2008; 283(16): 11024 - 11037. [Abstract] [Full Text] [PDF]

				D. B. Diep, M. Skaugen, Z. Salehian, H. Holo, and I. F. Nes Common mechanisms of target cell recognition and immunity for class II bacteriocins PNAS, February 13, 2007; 104(7): 2384 - 2389. [Abstract] [Full Text] [PDF]

				C. Bachler, K. Flukiger-Bruhwiler, P. Schneider, P. Bahler, and B. Erni From ATP as Substrate to ADP as Coenzyme: FUNCTIONAL EVOLUTION OF THE NUCLEOTIDE BINDING SUBUNIT OF DIHYDROXYACETONE KINASES J. Biol. Chem., May 6, 2005; 280(18): 18321 - 18325. [Abstract] [Full Text] [PDF]

				M. Aboulwafa and M. H. Saier Jr. Characterization of Soluble Enzyme II Complexes of the Escherichia coli Phosphotransferase System J. Bacteriol., December 15, 2004; 186(24): 8453 - 8462. [Abstract] [Full Text] [PDF]

				L. F. Garcia-Alles, K. Flukiger, J. Hewel, R. Gutknecht, C. Siebold, S. Schurch, and B. Erni Mechanism-based Inhibition of Enzyme I of the Escherichia coli Phosphotransferase System. CYSTEINE 502 IS AN ESSENTIAL RESIDUE J. Biol. Chem., February 22, 2002; 277(9): 6934 - 6942. [Abstract] [Full Text] [PDF]

				R. Gutknecht, K. Flukiger, R. Lanz, and B. Erni Mechanism of Phosphoryl Transfer in the Dimeric IIABMan Subunit of the Escherichia coli Mannose Transporter J. Biol. Chem., March 5, 1999; 274(10): 6091 - 6096. [Abstract] [Full Text] [PDF]

				R. Gutknecht, M. Manni, Q. Mao, and B. Erni The Glucose Transporter of Escherichia coli with Circularly Permuted Domains Is Active in Vivo and in Vitro J. Biol. Chem., October 2, 1998; 273(40): 25745 - 25750. [Abstract] [Full Text] [PDF]

				R. Gutknecht, R. Lanz, and B. Erni Mutational Analysis of Invariant Arginines in the IIABMan Subunit of the Escherichia coli Phosphotransferase System J. Biol. Chem., May 15, 1998; 273(20): 12234 - 12238. [Abstract] [Full Text] [PDF]

				R. Lanz and B. Erni The Glucose Transporter of the Escherichia coli Phosphotransferase System. MUTANT ANALYSIS OF THE INVARIANT ARGININES, HISTIDINES, AND DOMAIN LINKER J. Biol. Chem., May 15, 1998; 273(20): 12239 - 12243. [Abstract] [Full Text] [PDF]

				C. L. Bouma and S. Roseman Sugar Transport by the Marine Chitinolytic Bacterium Vibrio furnissii. MOLECULAR CLONING AND ANALYSIS OF THE MANNOSE/GLUCOSE PERMEASE J. Biol. Chem., December 27, 1996; 271(52): 33468 - 33475. [Abstract] [Full Text] [PDF]

				S. Mukhija and B. Erni Purification by Ni2+ Affinity Chromatography, and Functional Reconstitution of the Transporter for N-Acetylglucosamine of Escherichia coli J. Biol. Chem., June 21, 1996; 271(25): 14819 - 14824. [Abstract] [Full Text] [PDF]

				Q. Mao, T. Schunk, B. Gerber, and B. Erni A String of Enzymes, Purification and Characterization of a Fusion Protein Comprising the Four Subunits of the Glucose Phosphotransferase System of Escherichia coli J. Biol. Chem., August 4, 1995; 270(31): 18295 - 18300. [Abstract] [Full Text] [PDF]

				Q. Mao, R. G. Deeley, and S. P. C. Cole Functional Reconstitution of Substrate Transport by Purified Multidrug Resistance Protein MRP1 (ABCC1) in Phospholipid Vesicles J. Biol. Chem., October 27, 2000; 275(44): 34166 - 34172. [Abstract] [Full Text] [PDF]